Erythrocyte sedimentation rate control

ABSTRACT

A three-phase suspension suitable for use as an erythrocyte sedimentation rate control having the following three components: (1) a synthetic plasma base, (2) a high molecular weight polymer, and (3) mammalian red blood cells. The control is designed to allow the user to monitor the accuracy and precision of analytical methods for determining the sedimentation rate of human erythrocytes in whole blood specimens.

This application is a divisional of U.S. application Ser. No.08/538,959, filed on Oct. 4, 1995, now U.S. Pat. No. 5,888,822 issued onMar. 30, 1999. The priority of this prior application is expresslyclaimed and its disclosure is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to the measurement of erythrocyte sedimentationrate (ESR), and more particularly to a blood control standard for thequality control of the measurement of ESR.

BACKGROUND OF THE INVENTION

The ESR test measures the sedimentation rate of aggregated erythrocytesin plasma. The rate of sedimentation is an indirect means ofquantitating Rouleaux formation as well as red cell aggregation.Sedimentation occurs because the apparent surface/volume ratio of thered cells decreases and the denser Rouleaux overcome the buoyant forcesof the plasma and sink. Erythrocyte sedimentation depends upon aninterrelationship of a number of inherent biologic variables. Anythingthat increases the tendency to form Rouleaux or red cell aggregationwill accelerate the sedimentation rate. In vivo, the plasmaconcentrations of proteins and globulins as well as the shape of the redblood cells are the most important factors contributing to the ESR.

In most normal persons, sedimentation takes place slowly, but in avariety of disease states the rate is rapid and in some casesproportional to the severity of the disease. The ESR test has beenutilized as an indirect measure of these disease states. However, thetest is very non-specific in that values for "normal" ESR may beinfluenced by local conditions as well as the age and sex of thepatient. Nonetheless, the ESR test is an extremely common test whichplays a significant role in contemporary medical practice.

Westergren developed the technique of performing an ESR determination asdescribed in a paper published in 1924. See Alf Westergren, "Diesenkungscreaktion", Ergegn. Inn. Med. Kinderheilk., 26:577 (1924). Inthe Westergren method, a blood sample is obtained by venepuncture and isthoroughly mixed with a suitable anticoagulant. Because the proteins andglobulins in blood are unstable in vitro, at room temperature the testmust be set up within two hours, or at 4E C within 6 hours. Theblood-anticoagulant is thoroughly mixed by gentle repeated inversion anda clean dry standard Westergren-Katz tube is filled and adjusted to the`0` mark. The tube is then placed in a strictly vertical position underroom temperature conditions (18-25E C), not exposed to direct sunlightand free from vibrations and drafts. After a time period, usually onehour, the distance (x) from the bottom of the surface meniscus to thetop of the column of sedimenting red cells (where the full density isapparent), is read in mm and recorded as the ESR value. The result isexpressed as follows: `ESR (Westergren 1 hr)=×mm`. Variations in thematerials and methods are known, however, the basic technique isrelatively unchanged since its introduction.

Due to the manner in which ESR is measured, in addition to the biologicvariables certain identifiable environmental and technical factors mayinfluence the ESR test in misleading ways. For example, the followingfactors may affect the measurement of ESR:

Environmental Factors:

1. Temperature. The room temperature during the test could lead to amisleadingly high ESR (higher temperatures) or low ESR (lowertemperatures). Further, a variation of temperature during the test willalso lead to misleading results.

2. Vibration. Vibration or movement of the testing apparatus during thetest will result in misleading results.

Procedural Factors:

1. Positioning of tube. The correct or incorrect positioning of the tubeat a perpendicular angle will affect test results.

2. Delay prior to test. A delay in performing the test beyond 2 hours ofdrawing the blood sample will create ambiguous results.

3. Insertion of tube in reservoir (for modified Westergren procedures).Failure to fully insert the tube to the bottom of the reservoir incertain modified Westergren procedures will affect the test results.

4. Unfamiliarity or failing to follow manufacturer's directions willaffect test results.

Testing Materials Factors

1. Tube. Variations of the composition and/or length of the measurementtube will affect test results. For example, the use of glass vs. plastictubes in either a Wintrobe or Westergren procedure will lead tovariations in the observed sedimentation rate.

2. Anticoagulant. The anticoagulant used will affect test results.

3. Plasma. Changes in the plasma composition is a significant factordetermining the measured ESR.

There currently is no known commercial control by which the foregoing,and other, factors can be eliminated as sources affecting test results.Accordingly, a given ESR measurement can only be accepted as within arelatively large range of error. This decreases the significance of theESR test.

SUMMARY OF THE INVENTION

The present invention is a reference control designed to monitor theaccuracy and precision of analytical methods for determining thesedimentation rate of human erythrocytes in whole blood specimens. TheESR control comprises a three component colloidal/emulsion suspension:(1) a synthetic plasma base, (2) a polymer having a high molecularweight in the range of between 15,000-500,000, and (3) mammalian redblood cells. In the preferred embodiment, the polymer used is Dextran, apolysaccharide.

Dextran is known for its use as a plasma expander used for cellseparation, but is used for a different purpose in the ESR control ofthe present invention. Here, the addition of Dextran to the syntheticplasma base serves the function of behaving similar to an abnormalincrease of large plasma proteins such as Fibrinogen and Alpha 2macro-globulins in whole blood. It has been proposed that abnormalincreases in plasma protein concentrations cause an increase inaggregate formation resulting in an increased ESR. The Dextran andsynthetic plasma base is a stable suspension that maintains themorphology of the red cells for long periods of time to allow for acontrolled sedimentation of the cells. An ESR measurement using the ESRcontrol should fall within a fairly narrow, predictable range.Measurements outside this range would therefore indicate flaws in thetesting method.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A number of subheadings are provided in the following discussion inorder to provide organization and clarity.

1. Background and Theory

The ESR control of the present invention can be better understood ifcertain aspects of the phenomenon of sedimentation of erythrocytes inwhole blood are described. Accordingly, a discussion of erythrocytesedimentation and the theory of the ESR control are presented.

As previously discussed, the composition of the plasma is one of the twomost important factors in determining erythrocyte sedimentation rate(the other being the shape of the red blood cells). The components inthe plasma which create the Rouleaux effect and red cell aggregation arethe plasma proteins and colloids, particularly the Fibrinogen, Alpha 1and Alpha 2 globulins found in whole blood. It is known that allproteins affect the dielectric coefficient of plasma, but asymmetricalmacromolecules are oriented by the field and hence exert adisproportionately large effect. Therefore, as fibrinogen and gammaglobulin in plasma increase, they decrease the zeta potential ofsuspended red cells, permitting increased rouleaux formation and morerapid sedimentation rate. See Brian S. Bull and J. Douglas Brailsford,"The Zeta Sedimentation Ratio", Blood 1972:40:550. The ideal controlwould therefore incorporate these colloids and proteins. However, thechemistry of these components makes them unstable for any extendedperiod of time, and would therefore limit the useful life of a controlthat included these components.

The use of a high molecular weight polymer such as Dextran in asynthetic plasma base serves to mimic the function of the Fibrinogen,Alpha 1 and Alpha 2 globulins found in whole blood by creating theRouleaux effect. The two-phase Dextran/synthetic plasma suspensiontherefore behaves similarly to natural plasma with the advantage ofbeing a stable compound. Unlike the plasma proteins and colloids,Dextran is stable in synthetic plasma.

The second important factor in accelerated erythrocyte sedimentation isthe morphology of the cells. As fresh cells are allowed to sit in anEDTA or Sodium Citrate solution, they tend to crenate and manifest thephenomenon of anisocytosis and poikilocytosis along with exhibitingchanges in the electrical charges on the surfaces of the red cells. Eachof these changes tends to inhibit the sedimentation velocity. However,the Dextran/synthetic plasma base suspension has been found to maintainthe morphology of pre-treated, stabilized cells for long periods of timeto allow for a controlled precipitation of the erythrocytes. Thesynthetic plasma base which bathes the mammalian red cells appears tocause no variation of size and shape of the cells, thereby allowing thecells to maintain a constant morphology.

2. Components, Composition, and Method of Making

Turning now to the components and composition of the ESR control of thepresent invention, the control comprises a three componentcolloidal/emulsion suspension: (1) a synthetic plasma base, (2) apolymer having a high molecular weight substantially in the range ofbetween 15,000-500,000, and (3) pretreated, stabilized mammalian redblood cells. The three component colloidal/emulsion suspension isintended for use as a control for a given ESR test apparatus and method.

The ESR control is intended to exhibit the characteristics of a sampleof human blood for the purpose of performing an ESR test. The componentsof the ESR control are therefore formulated to mimic thesecharacteristics of human blood, such that there is provided a suspensionthat simulates an actual patient sample and that produces reproduciblesedimentation rates within a predictable range of values. A commerciallyuseful embodiment of the ESR control is further advantageouslyformulated to be stable for periods of prolonged storage.

The synthetic plasma base component of the ESR control is a carryingmedia that substitutes for plasma in natural blood and that maintains ahomeostatic environment for the other components of the ESR control,particularly the red blood cells. For use in the ESR control, thesynthetic plasma base comprises a carrying media that, in conjunctionwith the other components of the ESR control, produces predictable andreproducible red blood cell sedimentation rate values for a given ESRtesting apparatus and method. In commercial ESR control products, it ispreferable that the synthetic plasma base be formulated to stabilize thered blood cells and to maintain the morphology and specific gravity ofthe red blood cells, thereby conferring long-term stability on the ESRcontrol.

To produce predictable and reproducible sedimentation rates in the ESRcontrol, it has been found that Alsever's solution or a modifiedAlsever's solution-type synthetic plasma base is suitable for use as thesynthetic plasma base component. It has been found that an ESR controlhaving Alsever's solution as the synthetic plasma base component doesnot form a colloidal emulsion/suspension since the high molecular weightpolymer component tends to dissolve in the Alsever's solution, however,this occurrence does not significantly effect the performance of the ESRcontrol. An ESR control which relies substantially on Alsever's solutionas the synthetic plasma base will be suitable if the requisite stabilitydoes not exceed a few days. Alsever's solution is a transport media wellknown in the art, comprising 2.05 gm dextrose, 0.80 gm sodium citrate,0.42 gm sodium chloride, and 0.05 gm citric acid in 100 ml distilledwater. Alsever's solution is well known as a media having preservativeproperties and that is useful for suspending living cells or tissuesunder investigation in vitro. As noted, the use of Alsever's solution asthe synthetic plasma base component will provide an ESR control thatproduces predictable and reproducible ESR measurements, however, in anESR control product with extended shelf life, it is advantageous toprovide an altered formulation for the synthetic plasma base whichincludes components which are known to provide improved cell stabilityand prolonged shelf life.

For example, the addition of buffers to the synthetic plasma base hasbeen found to assist in maintenance of a relatively constant pH.Bactericides and fungicides may be added to assist in retarding theadverse effects of contamination prior to or during storage. Proteinstabilizers and cryoprotectants may be added to contribute to cellstability. Those skilled in the art will recognize that these and othermaterials may be added to the synthetic plasma base to provideadditional beneficial results while not inhibiting the performance ofthe ESR control.

Accordingly, as a practical matter it is preferable to include materialsin the synthetic plasma base that contribute to the long-term stabilityof the ESR control. To achieve this end, the synthetic plasma base isprovided with materials that maintain the morphology and integrity ofthe red blood cells to allow the red blood cells to mimic the behaviorof red blood cells in vivo, and to do so even after prolonged periods ofstorage. In relation to the interaction between the synthetic plasmabase and the red blood cells, the properties of a synthetic plasma basewhich make it suitable for use in a commercially practical ESR controlare the following: (1) the synthetic plasma base stabilizes the redblood cells in a manner that maintains the cell membranes in a dynamicstate, (2) the synthetic plasma base maintains the morphology of the redblood cells, and (3) the synthetic plasma base maintains the specificgravity of the red blood cells.

A preferred form of the synthetic plasma base for use in thecommercially practical ESR control is a synthetic plasma base that isprovided with one or more of the materials described above, such asantibiotics, antifungals, protein stabilizers, cryoprotectants, andbuffers. The synthetic plasma base is preferably formulated to exhibitcharacteristics similar to human plasma, such as specific gravity, pH,and potassium and sodium ion concentrations.

An example of a synthetic plasma base having the above-describedproperties which has been found to be suitable for use in the ESRcontrol is the synthetic plasma base having the formulation listed belowin Table 1. The synthetic plasma base having the formulation describedin Table 1 has been found to confer long term stability to the red bloodcells in the ESR control. Those skilled in the art will recognize thatother and further variations of this formulation will provide theproperties referred to above that make a solution suitable for use asthe synthetic plasma base of the ESR control.

                  TABLE 1                                                         ______________________________________                                        Synthetic Plasma Base For Use in ESR Control                                    Component                 Amount Per Batch                                  ______________________________________                                        Distilled Water         40       L                                              Reagent Alcohol 1,400 ml                                                      Sodium Chloride 40 gm                                                         Sodium Fluoride 30 gm                                                         Sodium Citrate 288 gm                                                         Citric Acid 20 gm                                                             Sodium Nitrate 200 gm                                                         3-N-morpholino propane sulfonic acid (MOPS) 80 gm                             Potassium ferrocyanide 24 gm                                                  Sodium hydroxide 12 gm                                                        Polyethylene glycol (M.W. Approx. 3500) 400 gm                                Polyethylene glycol (M.W. Approx. 7000) 2,400 gm                              Methyl paraben 40 gm                                                          Ethyl paraben 20 gm                                                           Bovine serum albumin (BSA) Fraction V 80 gm                                   Tetracycline 12 gm                                                            Streptomycin 20 gm                                                            Penicillin 20 gm                                                              Neomycin 12 gm                                                              NaOH or HCl             q.s. to pH 7.0".02                                      NaCl or Distilled Water q.s. to conductivity                                   10,300-10,600                                                              ______________________________________                                    

All of the chemicals listed above are available from Sigma Chemical, St.Louis, Mo. For best results, the components of the synthetic plasma baselisted in the table are added in the order listed. The completeformulation is then filtered through a 0.2 μ cartridge filter.

The high molecular weight polymer component of the ESR control isintended to contribute to sedimentation of the red blood cells in thesuspension by causing increased Rouleaux formation. In this way, thehigh molecular weight polymer functions similar to an abnormal increaseof the Fibrinogen, Alpha 1 and Alpha 2 globulins which perform the samefunction in whole blood as noted above. It has been found that a highdensity inert molecule that is physiologically compatible with red bloodcells will tend to increase the aggregation of red blood cells in such amanner. Increased aggregation of red blood cells is believed to becaused by the polymer component of the ESR control due to the samephysical and electrical mechanism by which the plasma proteins andcolloids contribute to red cell aggregation in whole blood.

In the preferred embodiment, Dextran having a molecular weight ofsubstantially between 15,000 and 500,000 is utilized as the highmolecular weight polymer, though those skilled in the art willappreciate that other high density inert molecules might alternativelybe used. Dextran is a polysaccharide having a chain-like structurecomprising a combination of certain polymers of glucose. It is producedfrom sucrose by Leuconostoc bacteria. Dextran is stable to heat andstorage and is soluble in water, making very viscous solutions. Dextranis known for its use as a blood plasma substitute or expander, andparticularly for its use in this regard for cell separations.

Dextran has been found to be a preferred material for use in the ESRcontrol because it causes Rouleaux formation while maintaining cellmembranes and cell morphologies intact. However, it is proposed that thefollowing additional polymers may be found to provide adequate resultsin the ESR control:

1. Ficoll (MW 70,000-400,000): A synthetic polymer made bycopolymerization of sucrose and epichlorhydrin that is widely used as adensity gradient centrifugation medium. It is also used as animmunologically inert carrier for low-molecular-weight haptens inimmunological studies.

2. Cellulose: A high-molecular-weight polysaccharide comprising longunbranched chains of (1,4)-linked β-D-glucose residues. Cellulose isfound in cell walls of higher plants and some fungi as microfibrils, inwhich the cellulose chains form crystalline micelles separated byregions of randomized amorphous cellulose.

3. Cyclodextrin: Any of a number of oligosaccharides based onglucopyrinose units that are linked to form a ring structure. Themolecule consists of an apolar, electron-rich, hydrophobic interior withexterior sites available for hydrophilic interactions at the entrancesto the internal cavity.

4. Agar: A complex polysaccharide produced by red algae. It contains thepolysaccharides agarose and agaropectin. Agar is used in foodmanufacture and as a matrix for the culture of microorganisms.

5. Agarose: A polysaccharide gum obtained from seaweed composed ofalternating (1,3)-linked D-galactose and (1,4)-linked3,6-anhydro-D-galactose residues, as well as small amounts of D-xylose.Some of the D-galactose units are methylated at C-6. Agarose is used asa gel medium in chromatography or electrophoresis.

6. Starch: A high-molecular-weight polysaccharide consisting largely ofD-glucose units linked through an α-(1,4)-link, forming a spiral chainwith only one terminal reducing moiety per chain. It consists of twofractions: amylose (25 percent) and amylopectin (75 percent). It is themajor storage carbohydrate in higher plants, where it accumulates in theform of grains.

7. Polyvinylpyrolidone (PVP) (MW 10,000-360,000).

8. Polyethylene glycol (MW 200-20,000).

9. Percoll: A colloidal PVP coated with silica, used for cell separationand for tissue cultures.

10. Dimethylpolysiloxane (MW 770-116,500).

Those skilled in the art will recognize that other and further polymersin addition to the polymers described above would be suitable for use inthe ESR control. The function of the high molecular weight polymercomponent of the ESR control is to increase red cell aggregation and tothereby contribute to red cell sedimentation. Accordingly, it isbelieved that any high density inert molecule that is physiologicallycompatible with red cells would be suitable for use in the ESR control.Those specific examples provided herein are intended to illustrate thetypes of molecules suitable for use, rather than to limit them.

The final component of the ESR control is mammalian red blood cells. Awide range of sizes of red blood cells are suitable for use in the ESRcontrol, and typically the cell sizes will range over a standardGaussian distribution curve having an MCV of about 85 cubic microns.This range of cell sizes is not critical, but is utilized in thepreferred embodiment because the range approximates that of normal cellspresent in a drawn sample of human blood. In the preferred embodiment,the mammalian red blood cells are pre-treated by performingapproximately three washings of the cells in the synthetic plasma baseover an approximately 21 day period. Pretreatment of the red blood cellsstabilizes the cells and causes the cells to maintain their morphologysuch that the ESR control is stable over an extended period of time.

The following is an example of a method of making an ESR control inaccordance with the present invention. As will be more fully discussedbelow, the molecular weights, concentrations, Hematocrit values andcompositions are intended for exemplary purposes only, and are in no wayintended to limit the scope of the present invention.

The method comprises the following:

1. Prepare a 3% solution of Dextran (MW 184,000) in the synthetic plasmabase. For example, add 30 grams of Dextran powder to 1000 ml ofsynthetic plasma base. The suspension should be mixed for at least 3hours on a rotating mixer or with a magnetic mixer. Visually confirmthat the powdered Dextran has dissolved.

2. Combine units of pre-treated red blood cells to approximate 1 liter.Centrifuge the units of pre-treated cells at 2000 rpm for 30 minutes at5-10E C. Extract as much of the supernatant necessary to yield a RBCcount of 900,000 to 1,000,000 per cubic millimeter. (If no cell counteris available, utilize a microhematrocrit centrifuge to verify aHematocrit of 80% or more.)

3. Utilizing a graduated cylinder, pour 200 ml of the concentratedpre-treated red blood cells and q.s. to 1000 ml of the 3%Dextran/synthetic plasma base mixture. (Note: Each time the 3%Dextran/synthetic plasma base mixture is used, it should be mixed byinversion, e.g. 10-15 times. This is a 2 phase suspension that willseparate upon standing.)

4. Perform a Spun Hematocrit on the three component mixture and verifythat it has a Spun Hematocrit of 20% " 1%. (Note: Each time thefinalized product is tested for Hematocrit readings, be sure to mix bygentle inversion, e.g. 10-15 times. This is a 3 phase suspension thatwill separate upon standing.)

5. Pour the three phase suspension into a container which willaccommodate greater than one liter. Make adjustments to the mixture bythe addition of concentrated red blood cells or 3% Dextran/syntheticplasma base mixture. This is a trial and error step that may requireseveral additions to achieve the desired 20% Hematocrit value.

6. Keep the finished mixture refrigerated for 24 hours and verify thatthe Hematocrit is stabilized at 20% " 1%.

The ESR control produced by the foregoing process has been found toprovide ESR readings in a "High Normal" range. It has also been foundthat an "Abnormal" range of ESR readings are observed where theforegoing procedure is altered by changing the amount of concentratedpretreated red blood cells added to 1000 ml of the 3% Dextran/syntheticplasma base mixture in step 3 from 200 ml to 150 ml, and then performinga Spun Hematocrit on the mixture in step 4 to obtain a Spun Hematocritof 15% " 1%. Other levels of control are obviously possible by simplyvarying the amount of concentrated red blood cells used in step 3 toobtain different Spun Hematocrit values of the final product.

The ESR control produced by the foregoing method is a stable three-phasesuspension which behaves in a fairly predictable manner when used as acontrol in an ESR test. For example, Tables 2 and 3 below list theresults (in mm) of precision studies performed using the two assaylevels, "High Normal" and "Abnormal", described above. Further, thetables provide results of ESR tests using the two levels of control withseveral manufacturer's testing apparatuses, two different testingmethods (Westergren and Wintrobe), and two different tube materials(glass and plastic):

                  TABLE 2                                                         ______________________________________                                        ESR Readings of "High Normal" Assay Level                                       (Tests run at temperatures ranging 19 E C.-21 E C.)                                     LP                                                                  MFR: Italiana Baxter Baxter Ulster Baxter                                     METHOD: Westerg. Westerg. Westerg. Wintrobe Wintrobe                          MTL: Plastic Plastic Glass Plastic Glass                                    ______________________________________                                                10       7        15     11     4                                        9 8 14 10  5                                                                  12  8 12 7 5                                                                  6 3 12 6 5                                                                    11  3 14 10  6                                                                10  6 13 3 2                                                                  3 4 12 6 3                                                                    5 4 13 2 3                                                                    5 9 14 7 4                                                                    9 7 12 3 4                                                                    9 6 13 4 4                                                                    8 4 13 3 4                                                                    10  5 10 5 4                                                                  7 3 13 4 3                                                                    11  8 12 3 4                                                                  6 3 16 3 2                                                                    8 4 12 6 3                                                                    8 5 16 3 3                                                                    9 3 14 6 3                                                                    13  6 15 8 2                                                                  3 8 15 8 4                                                                    10  8 10 6 4                                                                  5 5 13 6 3                                                                    3 5 11 3 3                                                                    5 4 15 5 4                                                                    5 5 15 2 7                                                                    3 6 13 6 7                                                                    2 6 13 3 4                                                                    9 3  8 5 4                                                                    8 3 12 7 6                                                                    12  6 12 3 3                                                                  7 2  7 2 3                                                                    4 7 12  4                                                                     9 6 13  5                                                                     6 7 11  6                                                                     5 5 15  5                                                                     5 5 12  4                                                                     9 4 14  3                                                                     10  2 11  5                                                                   7 6 14                                                                        4 8                                                                           4 4                                                                            8                                                                             6                                                                           NUMBER: 42  44  40 32  39                                                     MEAN: 7 5 13 5 4                                                              S.D.: 3 2  2 2 1                                                              C.V.:   8.3   3.5   3.8   6.0   1.6                                         ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        ESR Readings of "Abnormal" Assay Level                                          (Tests run at temperatures ranging 19 E C.-21 E C.)                                     LP                                                                  MFR: Italiana Baxter Baxter Ulster Baxter                                     METHOD: Westerg. Westerg. Westerg. Wintrobe Wintrobe                          MTL: Plastic Plastic Glass Plastic Glass                                    ______________________________________                                                24       24       32     26     24                                       23 26 34 22 26                                                                24 24 33 27 26                                                                31 27 24 27 22                                                                30 26 25 26 24                                                                32 23 27 23 23                                                                33 20 29 22 28                                                                32 25 29 23 24                                                                30 23 30 25 25                                                                30 24 31 26 29                                                                36 26 32 27 24                                                                29 23 30 28 27                                                                24 25 33 24 25                                                                28 24 30 25 27                                                                20 27 32 29 26                                                                28 23 24 27 20                                                                22 29 28 25 25                                                                18 27 31 25 22                                                                27 26 28 28 27                                                                24 30 27 29 25                                                                25 31 29 24 31                                                                25 22 31 25 30                                                                25 25 32 21 31                                                                25 23 34 28 32                                                                24 30 26 21 25                                                                25 27 27 25 24                                                                27 24 32 21 27                                                                22 26 31 25 21                                                                25 25 29 23 26                                                                25 24 25 25 27                                                                22 25 31 25 19                                                                23 17 30 27 22                                                                28 25 29 34 24                                                                25 25 27 25 24                                                                23 26 27 25 22                                                                25 23 25 26 26                                                                27 22 31 26 20                                                                24 21 34 24 22                                                                25 22 30  23                                                                  28 25 21  23                                                                  25 22 21  25                                                                  25 20 22  22                                                                   15                                                                            25                                                                            27                                                                            22                                                                            22                                                                            26                                                                            19                                                                            22                                                                            26                                                                            21                                                                          NUMBER: 42 52 42 38 42                                                        MEAN: 26 24 29 25 25                                                          S.D.:  4  3  3  3  3                                                          C.V.:   13.0   9.2   11.9   6.5   9.0                                       ______________________________________                                    

Using components having the molecular weight, concentrations, Hematocritvalues and compositions in the method of the preceding examples willproduce an ESR control in accordance with the present invention.However, significant departures from those values are possible whilestill remaining within the scope of the present invention. For example,in the following three examples, in which the Hematocrit value, Dextranconcentration, and Dextran molecular weight are all varied, it isbelieved that the resulting suspensions will all produce an ESR controlthat provides an ESR reading of about 30 for a given test apparatus:

    ______________________________________                                                                          ESTIMATED                                     HEMATOCRIT DEXTRAN DEXTRAN APPROX.                                            VALUE CONCENTRATION MW ESR VALUE                                            ______________________________________                                        30        8%            280,000   30                                            25 5% 240,000 30                                                              15 3% 140,000 30                                                            ______________________________________                                    

The purpose of the preceding examples is to demonstrate that it is therelative values of the Hematocrit, Dextran concentration and Dextranmolecular weight utilized in the ESR control, rather than the valuesthemselves, that are important to the effectiveness of the suspension asan ESR control. The purpose of the ESR control is to produce results ina predictable range of ESR values; accordingly, as long as thecomposition is such that consistent ESR values are obtained, thecomposition is useful as an ESR control. Those skilled in the art willrecognize that other and further variations of the values listed aboveare possible.

While the above description contains many specificities, these shouldnot be construed as limitations on the scope of the invention, butrather as an exemplification of a preferred embodiment thereof. Othervariations are possible.

Accordingly, the scope of the present invention should be determined notby the embodiments illustrated above, but by the appended claims andtheir legal equivalents.

What is claimed is:
 1. A method for preparing an erythrocytesedimentation rate control comprising the steps of:a. preparing asuspension comprising an aggregating agent in a synthetic plasma base;and b. adding mammalian red blood cells to the suspension of step a. toobtain a suspension having a hematocrit value, wherein sedimentation ofthe red blood cells within the suspension takes place at a substantiallyreproducible rate.
 2. The method of claim 1, wherein said syntheticplasma base includes protein stabilizers.
 3. The method of claim 1,wherein said synthetic plasma base includes cryoprotectants.
 4. Themethod of claim 1, wherein said synthetic plasma base comprisesantibiotics, antifungals, protein stabilizers, cryoprotectants andbuffers.
 5. The method of claim 1, wherein said synthetic plasma basecomprises tetracycline, methyl paraben, albumin, glycol, and3-N-morpholino propane sulfonic acid.
 6. The method of claim 1, whereinsaid synthetic plasma base comprises about 0.3 g/l tetracycline, about1.0 g/l methyl paraben, about 2.0 g/l albumin, about 70.0 g/l glycol,about 2.0 g/l 3-N-morpholino propane sulfonic acid, and q.s to 1 literdistilled water.
 7. The method of claim 1, wherein said synthetic plasmabase further comprises about 35 ml/l of reagent alcohol comprising oneor more from the group consisting of methanol, ethanol and isopropanol.8. The method of claim 1, wherein the suspension further comprises about0.60 g/l potassium ferrocyanide.
 9. The method of claim 1, wherein saidaggregating agent is a chemically inert molecule that is physiologicallycompatible with red blood cells.
 10. The method of claim 1, wherein saidaggregating agent is a polysaccharide.
 11. The method of claim 1,wherein said aggregating agent is one or more from the group consistingof Dextran, Ficoll, Cellulose, Cyclodextrin, Agar, Agarose, Starch,Polyvinylpyrol idone, Polyethylene Glycol, Percoll, andDimethylpolysiloxane.
 12. The method of claim 1, wherein saidaggregating agent is dextran.
 13. The method of claim 12, wherein saiddextran has a molecular weight substantially in the range between 15,000to 500,000.
 14. The method of claim 12, wherein said dextran has amolecular weight substantially in the range between 125,000 to 275,000.15. The method of claim 12, wherein said dextran has a concentration ofbetween about 30-80 g/l in said synthetic plasma base.
 16. The method ofclaim 1, wherein said mammalian red blood cells are pre-treated bywashing in said synthetic plasma base.
 17. The method of claim 1,wherein the sizes of said mammalian red blood cells range over astandard Gaussian distribution curve with a mean cell volume of about 85cubic microns.
 18. The method of claim 1, wherein said mammalian redblood cells have a mean cell volume of between 80-90 microns.
 19. Themethod of claim 1, wherein the hematocrit value is between about 15% toabout 30%.
 20. The method of claim 1, wherein the hematocrit value isabout 15%.
 21. The method of claim 1, wherein the hematocrit value isabout 20%.